Structural basis for antibody recognition of vulnerable epitopes on Nipah virus F protein

Nipah virus (NiV) is a pathogenic paramyxovirus that causes fatal encephalitis in humans. Two envelope glycoproteins, the attachment protein (G/RBP) and fusion protein (F), facilitate entry into host cells. Due to its vital role, NiV F presents an attractive target for developing vaccines and therapeutics. Several neutralization-sensitive epitopes on the NiV F apex have been described, however the antigenicity of most of the F protein’s surface remains uncharacterized. Here, we immunize mice with prefusion-stabilized NiV F and isolate ten monoclonal antibodies that neutralize pseudotyped virus. Cryo-electron microscopy reveals eight neutralization-sensitive epitopes on NiV F, four of which have not previously been described. Novel sites span the lateral and basal faces of NiV F, expanding the known library of vulnerable epitopes. Seven of ten antibodies bind the Hendra virus (HeV) F protein. Multiple sequence alignment suggests that some of these newly identified neutralizing antibodies may also bind F proteins across the Henipavirus genus. This work identifies new epitopes as targets for therapeutics, provides a molecular basis for NiV neutralization, and lays a foundation for development of new cross-reactive antibodies targeting Henipavirus F proteins.


nature portfolio | reporting summary
March 2021

Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy

Human research participants
Policy information about studies involving human research participants and Sex and Gender in Research.
Reporting on sex and gender Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size

Data exclusions
Replication Randomization Blinding Negative-stain EM: Manual correction using EMANS, reference-free 2D classifications were performed with Relion 1.4, FACS: FlowJo software version 9.9.4 (Tree Star, Inc), Neutralization assay: IC80 is calculated by curve fitting and nonlinear regression (Log(agonist) vs normal response (variable slope) EC) using GraphPad Prism v8,Biolayer interferometry: Octet Data Analysis v12.0.2.3, Percent competition (PC) of analyte mAbs binding to competitor-bound NiV prefusion F was determined using the equation : PC = 100 -[(analyte mAb binding in the presence of competitor mAb) / (analyte mAb binding in the absence of competitor mAb)] x 100, CryoEM: Movies collected using SerialEM, Motion correction and CTF-estimation performed in WARP or cryoSPARC Live, Micrographs imported into cryoSPARC for particle picking, 2D classification, ab initio 3D reconstruction and 3D refinement, Homology models for Fabs were generated using ABodyBuilder, Initial models were docked into the cryo-EM maps using Chimera, Complementarity-determining loops were built manually in Coot, Models were iteratively refined using Coot, Phenix and ISOLDE, SPR: Data were double reference-subtracted and fit to a 1:1 binding model using Biacore Evaluation Software Structural models are deposited in the protein data bank (PDB, https://www.rcsb.org/) and are scheduled to be released upon publication of this paper. NA NA NA NA 10 female CB6F1/J mice were immunized, spleens from 4 randomly selected mice were pooled for B-cell sorting/antibody isolation.
No data was excluded.
Initial screening/characterization of identified antibodies included binding analysis, neutralization assays and competition binding studies. All studies or relevant portions of studies (i.e. competition binding studies with identified neutralizing antibodies competing with other neutralizing antibodies) were repeated with comparable results at least twice.
Animals were randomly allocated to immunization groups at start of study.
No blinding to immunization was used. This is a non-clinical study with data collection and analyses relying on objective measures.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. VSV G antibody was validated in infection assays of VSV"G-G-luc stock and NiV F/G VSV"G-luc stock preparations, 5B3 antibody was validated by binding assays (to pre-F and post-F designs), pseudovirus neutralization assays and negative stain EM bound to Nipah F protein, antibody panel used for B-cell sorting were titrated using FACS as described in the methods and shown in Supplementary Figure 1 with final dilution for sort selected highlighted in Supplementary Figure 1B.

Materials
Vero E6 cells were purchased from ATCC (VERO C1008, clone E6, catalog number -CRL-1586) Cells lines were not authenticated. They were purchased directly from vendor and maintained and frozen according to manufacturer's instructions.
Cell lines were not tested for mycoplasma contamination.
Cell lines were not tested for mycoplasma contamination.
CB6F1/J mice from Jackson Laboratory, female, all mice were 6-8 weeks old at start of vaccination. Mice were maintained at 72°F +/-5°F, relative humidity of 30-70% (typically 33-40%) on a 12h light/dark cycle with food and water ad libitum.
No wild animals were used in this study.
Only female mice were used in this study. Male mice are more aggressive than female mice. Subsequent studies in ferrets showed no variability in immunogenicity between the sexes.